# US 2014/0193468 A1 · Methods for preparation of neo-cartilage constructs

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- Number: US 2014/0193468 A1
- Document ID: US20140193468A1
- Status: Published application
- Kind: A1
- Application number: 14/208,931
- Filed: 2014-03-13
- Published: 2014-07-10
- Earliest claimed: 2001-03-23
- Assignees: Histogenics Corporation
- Applicants: Histogenics Corporation
- Inventors: Laurence J. B. Tarrant; Sonya Shortkroff; Shuichi Mizuno; Akihiko Kusanagi; Toshimasa Tokuno; Robert Lane Smith
- Web page: https://patentgrep.com/patent/US20140193468A1

## CPC

- A61K 38/39: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
- A61F 2/28: Bones
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 38/1825: {Fibroblast growth factor [FGF]}
- A61K 38/1875: {Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor}
- A61K 38/19: Cytokines; Lymphokines; Interferons
- A61K 38/30: Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2 {(insulin-like growth factor binding protein A61K38/1754)}
- A61L 27/24: Collagen
- A61L 27/38: {containing added animal cells (organs or tissue containing native cells A61L27/36)}
- A61L 27/3817: {Cartilage-forming cells, e.g. pre-chondrocytes}
- A61L 27/3834: {Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells}
- A61L 27/3852: {Cartilage, e.g. meniscus}
- A61L 27/3895: {using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions}
- A61L 27/54: Biologically active materials, e.g. therapeutic substances {(A61L27/227 takes precedence)}
- A61L 27/56: Porous materials, {e.g. foams or sponges}
- C12N 5/0655: {Chondrocytes; Cartilage}
- A61F 2310/00365: Proteins; Polypeptides; Degradation products thereof
- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- A61L 2300/414: Growth factors
- A61L 2430/06: for cartilage reconstruction, e.g. meniscus
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2533/54: Collagen; Gelatin
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61F: FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS (dental prosthetics A61C)
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention generally relates to systems (i.e. constructs) for repairing cartilage and methods for preparing the same that introduce a bioactive agent into a culture medium, suspension, scaffold, solution incorporated into the pores of the scaffold, or combinations thereof. The introduction of a bioactive agent promotes production of neo-cartilage (i.e. immature hyaline cartilage) in the system, both ex-vivo and in-vivo.

## Claims

### Claim 1

A system for repairing cartilage, the system comprising
an acellular collagen matrix comprising a plurality of pores, and
a solution disposed within the pores, said solution being treated with at least one bone inducing agent,
wherein said acellular matrix comprising the solution is lyophilized.

### Claim 2 (depends on 1)

The system of claim 1, wherein said solution is a collagenous solution.

### Claim 3 (depends on 1)

The system of claim 1, wherein the at least one bone inducing agent is selected from the group consisting of a growth factor, a cytokine, a matrix remodeling enzyme, a matrix metalloproteinase, an aggrecanase, a cathepsin, demineralized bone powder, calcium phosphate, hydroxyapatite, organoapatite, titanium oxide, poly-L-lactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a combination thereof.

### Claim 4 (depends on 3)

The system of claim 3, wherein said growth factor is a fibroblast growth factor (FGF), a bone morphogenic protein (BMP), insulin growth factor (IGF), transforming growth factor beta (TGF-B), or a combination thereof.

### Claim 5 (depends on 4)

The system of claim 4, wherein said fibroblast growth factor is FGF2, FGF4, FGF9, FGF18, or variants thereof.

### Claim 6 (depends on 5)

The system of claim 5, wherein said fibroblast growth factor is FGF2v1.

### Claim 7 (depends on 6)

The system of claim 6, wherein said FGF2v1 comprises a nucleotide sequence of SEQ ID NO: 1.

### Claim 8 (depends on 1)

The system of claim 1, further comprising a plurality of allogeneic or syngeneic cells disposed within the plurality of pores of said acellular collagen matrix.

### Claim 9 (depends on 8)

The system of claim 8, wherein said allogeneic or syngeneic cells comprise stem cells or bone marrow aspirate.

### Claim 10 (depends on 9)

The system of claim 9, wherein the stem cells are adult stem cells, mesenchymal stem cells, peripheral blood stem cells, induced pluripotent stem cells, or any combination thereof.

### Claim 11 (depends on 1)

The system of claim 1, wherein said acellular collagen matrix is a biodegradable collagenous sponge, a honeycomb or honeycomb-like matrix, a collagenous porous scaffold, or a thermo-reversible gelation hydrogel (TRGH).

### Claim 12 (depends on 1)

The system of claim 1, wherein said acellular collagen matrix is prepared from a compound selected from the group consisting of a Type I collagen, a Type II collagen, a Type IV collagen, gelatin, agarose, collagen containing proteoglycan, collagen containing glycosaminoglycan, collagen containing glycoprotein, and a combination thereof.

### Claim 13 (depends on 1)

The system of claim 1, wherein said plurality of pores comprise a pore size ranging from 50 uM to 500 uM.

### Claim 14

A method of repairing cartilage in a subject, said method comprising the steps of:
providing an acellular collagen matrix comprising a plurality of pores,
soaking said matrix in a solution to dispose the solution within said plurality of pores, wherein the solution was treated with at least one bone inducing agent prior to soaking;
lyophilizing the acellular matrix comprising the solution; and
implanting the lyophilized acellular collagen matrix into a cartilage lesion in said subject.

### Claim 15 (depends on 14)

The method of claim 14, wherein the at least one bone inducing agent is selected from the group consisting of a growth factor, a cytokine, a matrix remodeling enzyme, a matrix metalloproteinase, an aggrecanase, a cathepsin, demineralized bone powder, calcium phosphate, hydroxyapatite, organoapatite, titanium oxide, poly-L-lactic acid or a copolymer thereof, polyglycolic acid or a copolymer thereof, and a combination thereof.

### Claim 16 (depends on 15)

The method of claim 15, wherein said growth factor is a fibroblast growth factor (FGF), a bone morphogenic protein (BMP), insulin growth factor (IGF), transforming growth factor beta (TGF-B), or a combination thereof.

### Claim 17 (depends on 16)

The method of claim 16, wherein said fibroblast growth factor is FGF2, FGF4, FGF9, FGF18, variants thereof.

### Claim 18 (depends on 17)

The method of claim 17, wherein said fibroblast growth factor is FGF2v1.

### Claim 19 (depends on 18)

The method of claim 18, wherein said FGF2v1 comprises a nucleotide sequence of SEQ ID NO: 1.

### Claim 20 (depends on 14)

The method of claim 14, further comprising the steps of
seeding said lyophilized acellular collagen matrix with a plurality allogeneic or syngeneic cells, and
culturing the seeded matrix ex vivo under conditions sufficient for inducing cellular growth and differentiation,
wherein said steps are performed prior to implantation of said seeded matrix into the cartilage lesion in the subject.

### Claim 21 (depends on 20)

The method of claim 20, wherein said allogeneic or syngeneic cells comprise stem cells or bone marrow aspirate.

### Claim 22 (depends on 21)

The method of claim 21, wherein said stem cells are adult stem cells, mesenchymal stem cells, peripheral blood stem cells, induced pluripotent stem cells, or any combination thereof.

### Claim 23 (depends on 20)

The method of claim 20, wherein said culture conditions include applying hydrostatic pressure to the seeded implant.

### Claim 24 (depends on 20)

The method of claim 20, wherein said culture conditions do not include the application of hydrostatic pressure to the seeded implant.

### Claim 25 (depends on 14)

The method of claim 14, wherein a tissue sealant is used to implant the acellular matrix into the cartilage lesion in said subject.

### Claim 26 (depends on 25)

The method of claim 25, wherein said tissue sealant is deposited into the cartilage before the acellular matrix is implanted therein.

### Claim 27 (depends on 25)

The method of claim 25, wherein said tissue sealant is deposited over the acellular matrix after implantation into the cartilage of said subject.

### Claim 28 (depends on 25)

The method of claim 25, wherein said tissue sealant is selected from the group consisting of gelatin, a copolymer of polyethylene glycol and poly-lactide or poly-glycolide, periodate-oxidized gelatin, 4-armed pentaerythritol thiol and a polyethylene glycol diacrylate, 4-armed tetra-succinimidyl ester or tetra-thiol derivatized PEG, photo-polymerizable polyethylene glycol-co-poly(.alpha.-hydroxy acid) diacrylate macromer, 4-armed polyethylene glycol derivatized with succinimidyl ester and thiol further cross-linked with methylated collagen, derivatized polyethylene glycol (PEG), polyethylene glycol (PEG) cross-linked with alkylated collagen, tetra-hydrosuccinimidyl or tetra-thiol derivatized PEG, PEG cross-linked with methylated collagen, and a combination thereof.